PROJECTSUMMARY/ABSTRACT Lysosomescontrolasubstantialpartofcellularmetabolismbyactingasthemaincatabolichubofthecelland servingasaplatformfortheintegrationofnumeroussignalsthatmodulatecelldeath,growthandproliferation. Mostlysosomalfunctionsrelyonasetofmorethan50acidhydrolasesthatdegradeawidevarietyof macromolecules.Lysosomalenzymesaretraffickedtothelysosomeintwostages:transportofthenewly synthesizedproteinsfromtheendoplasmicreticulum(ER)totheGolgicomplex,andtheirsubsequent receptor-assistedtransferfromtheGolgitoendolysosomalcompartments.Howlysosomalenzymesare transportedfromtheERtotheGolgicomplexisunknownand,toourknowledge,thesimplemodelofabulk, unregulatedtransportationhasneverbeenquestioned.WehaveidentifiedtwocandidateERreceptors,CLN6 andCLN8,whosedeficiencyresultsinalteredmaturationoflysosomalenzymesandlysosomalstorage disorder-likediseases.WeproposetostudyhowCLN6andCLN8functioninthepathwayofmaturationof lysosomalenzymes.First,wewilltestthehypothesisthatCLN6andCLN8directlyinteractwithlysosomal enzymesandthatsuchinteractionisdisruptedbydisease-associatedmutationsoneitherCLN6/CLN8oron thesurfaceoflysosomalenzymes(Aim1).Second,wewillexaminethetraffickingandmaturationofnewly synthesizedlysosomalenzymestoidentifytheexactstepthatisdisruptedbyCLN6andCLN8deficiency.We willalsodefineCLN6andCLN8functionsinvivobycarryingoutdetailedtissue-specificanalysesoflysosomal compositioninCLN6-andCLN8-deficientmouselinesbyLC-MS/MS-basedproteomics.Tothisaim,wehave generatedaknock-inLamp1FLAGmouselinetoefficientlyisolatelysosomesfromthedesiredtissues(Aim2). Third,wewillidentifytheproteindomainsandmotifsthatareinvolvedinCLN6/CLN8interactionandthatdirect theirsortingacrossthecompartmentsoftheearlysecretorypathwayviaCOP-coatedvesicles(Aim3).Wewill accomplishourgoalswithamulti-disciplinaryapproachthatusesthetoolsofbiochemistry,molecularbiology, cellbiologyandmouseengineeringandwewillalsodevelopanewmethodofinvivolysosomeisolationfrom mousetissues.Ourresultsarelikelytohaveimportantconsequencesforourunderstandingofthe mechanismsgoverninglysosomalbiogenesisandofthemolecularpathogenesisofnumeroushuman diseases.Someoftheregulatorymechanismsweuncovermayserveinthefutureastargetsformodulating lysosomalbiogenesisindiseasesresultingfromimpairedlysosomalfunctionorinconditions,suchascertain typesofcancer,thatarecharacterizedbyaberrantorunrestrictedlysosomalactivation.